Leek (Allium ampeloprasum) belongs to the Alliaceae family. It is used as a vegetable and is grown worldwide, and in Europe it is an important commercial crop. Several factors make leek a difficult crop to improve (review Currah 1986). First it is a biannual crop, which means that in the first year the plant develops and after vernalisation in the winter it will flower in the second year.
Leek is also subject to severe inbreeding depression. The frequency of recessive lethal and deleterious genes is high. Already after one generation of selfing severe losses of vigor and seed production can be observed (Schweisguth 1970). As a consequence of the inbreeding depression, positive mass selection has been the most important way of improvement. Leek is an outbreeding species, but self-pollination does occur and frequencies up to 20% have been found in commercial cultivars (Beringer and Buret 1967). The open pollinated cultivars are highly heterogeneous. One of the factors contributing to this high heterogeneity is this high percentage of selfing, which results in inbreeding depression.
The cultivated leek is tetraploid (2n=4x=32), which makes breeding complicated. Most authors agree that leek is an autotetraploid, because for most of the genes leek shows tetrasomic inheritance (Schweisguth 1970, Beringer and Buret 1967). Objectives for leek breeding include improvement of winter-hardiness, a long shaft length, absence of bulbing, resistance to bolting, upright habitat, dark-green leaves and easy to peel. In addition to these improvements in quality, breeders also aim for improvement in yield, uniformity and resistance to pests and diseases.
Nowadays most breeding programs develop F1 hybrid cultivars, which have considerable benefits with regard to open-pollinated (OP) cultivars used before. Advantages of production of F1 hybrids are improvement of uniformity of the crop and better exploitation of heterosis for several traits, i.e. yield and faster fixation of desirable traits. Schweisguth (1970) has demonstrated heterosis in leek by making experimental hybrids between inbreds. These hybrids gave yields greater than the best open-pollinated cultivar and were more uniform.
To maintain the uniformity of the crop, prevention of selfing in hybrid seed production is needed. For some crops the selfing may be prevented by emasculation of the flowers, like is done for tomato and sweet pepper. In normal leek plants, however, the flowers are very small and there are many flowers on one flower head, which makes it impossible to emasculate by hand. Therefore, for the production of hybrids in leek it is advantageous to use plants that are male sterile.
Plants can be made sterile by genetic modification (GM). WO99/23233; Mogen Int. describes the production of male sterile plants by introducing recombinant DNA expressing trehalose phosphate phosphatase (TPP) in the plant. From the examples it can be seen that when the plant is transgenic for the TPP gene, it displays a male sterile phenotype. Tobacco, lettuce, and Arabidopsis are transformed with TPP. No examples in leek are given. In many countries, including most European countries, GM plants are not allowed and thus plants that are male sterile by transformation are not accepted in these countries.
Male sterility may be obtained by a non-GM route. One system for male sterility is based on cytoplasmic male sterility, like used in onions (Allium cepa). Many experiments and projects have been done to find or introduce this kind of male sterility into leek (Silvertand 1995, Buitenveld 1998). Some have been trying to introduce cytoplasm from other species like onion or Allium galanthum by conventional crossing or somatic hybridization. In WO2010/007059 cytoplasmic male sterility from garlic (Allium sativum) was introduced in leek plants. However, cytoplasmic male sterility (CMS) has a number of disadvantages including increased disease susceptibility, breakdown of sterility under certain conditions, the need to develop a set of CMS- and maintainer lines that are genetically the same, except for the cytoplasm (isogenic lines). This is necessary to multiply the CMS-line by seed. The expression of CMS can be complicated by the presence of restorer genes in the nucleus. Restorer genes are genes that can suppress the male sterile effect of the cytoplasm and are incorporated into the male parent to restore pollen fertility. Good maintainer lines need to be developed without restorer genes. In case of tetraploid inheritance in leek this is a complicated task, which can only be achieved by inbreeding the maintainer lines and selecting the ones without restorer. Inbreeding may then lead to strong inbreeding depression. When restorer genes are introduced, they may be linked to undesirable traits.
Another system for male sterility is nuclear encoded male sterility (NMS). This male sterility is controlled by nuclear genes. For NMS it is impossible to develop an isogenic set of NMS-line and maintainer line to multiply the NMS-line by seed, because the offspring of the NMS-line will always segregate for sterility. This means that the male sterile parent must be maintained vegetatively. This is now possible by using tissue culture or bulbils (little bulbs) produced in the flower head (Silvertand 1995). The progeny is a clone of the original sterile plant and shows the same traits. For NMS there is no need for development of sets of NMS- and maintainer lines, in contrast to CMS.
The nuclear male sterility as currently being used in modern hybrids has most likely a recessive nature. The assumption has been made that one or two recessive genes are responsible for the male sterility trait. Silvertand (1996) reports a percentage of 0.4% of naturally occurring male sterile plants, found after screening open pollinated seed productions in Italy.
If one wants to introduce recessive male sterility in another desired leek plant one has to cross the desired leek plant with a source for genetic male sterility. The heterozygous F1-generation produced there from has to be selfed and in the F2-generation a segregation for male sterility can be found. Assuming 1 recessive gene (ms), 1/36 of the F2-population shows the desired trait (Briggs & Knowles, 1967).
In a biannual crop like leek, one generation of backcrossing with a recessive male sterility trait costs 4 years: one generation of 2 years to make a cross between a sterile and fertile plant. The F1 plants are all fertile as the sterility is recessive in this case. Therefore another crossing step is needed which is often the selfing of the F1 to produce an F2, which takes another 2 years for a biannual crop. The F2 generation has to be grown until flowering because the distinction between male fertile and male sterile cannot be made until flowering. In the F2 generation a high amount of plants must be checked in order to find a male sterile plant because only 1/36=3% is sterile. If one also wants to have the possibility to select between male sterile plants to find a better plant with other desired traits, the F2 population has to be even bigger. The second cycle of backcross takes again 4 years because there will be no male sterile plants in the F1 because of the recessive nature of the male sterility trait. In order to convert a male fertile line into a similar male sterile line about 5 backcrosses are needed in a conventional backcross programs. This will take about 20 years for a tetraploid breeding crop like leek with a recessive male sterility trait.
It would be advantageous to have a dominant male sterility trait, because the male sterility trait would already appear in the F1 generation. This would reduce the stabile conversion into a male sterile line to about 10 years, instead of 20. In addition, for a dominant male sterile trait, in the F1 generation about 50% of the plants would be male sterile, instead of only 3% in the F2 generation of recessive male sterility. However, in order to maintain male sterility human intervention is required.
It is therefore an object of the present invention to provide a leek plant that possesses a dominant male sterility trait. Another object of the present invention is to identify genetic markers that are linked to this dominant male sterility trait. Yet another object of the present invention is to make hybrid leek plants and seeds with the dominant male sterility trait. In addition another object of the invention is to provide a method for selecting for dominant male sterile leek plants by using the genetic markers, as well as leek producing method using selection based on the genetic markers for dominant sterility.
One or more of these objectives are met by the present invention. The present invention provides a nuclear encoded male sterile leek plant wherein the male sterility trait is dominant. These male sterile leek plants produce about 50% male sterile F1 offspring. The male sterile leek plants of the present invention are used to produce F1 hybrid leek plant seeds. The seeds from the dominant male sterile leek plant are deposited at NCIMB Ltd, Aberdeen under number NCIMB 41699.
In addition, the present invention provides genetic markers that are linked to the dominant male sterility trait. Furthermore methods are provided to use the genetic marker in selection for male sterile leek plants.